We isolated DNA and genomic sequences encoding the human reduced folate carrier, which is involved in the transport of reduced folates and antifolates (methotrexates) into cells. We are now studying the regulation of RFC gene expression. We have begun to study the use of recombinant adenoviruses and adeno-associated viruses for gene therapy of cancer. Recombinant vectors have been constructed using transgenes that: 1) induce apoptosis (wild type p53); 2) inhibit cell proliferation (cyclin kinase inhibitors: p21/WAF1/CIP1, p16/INK4, p27/kip1); 3) converts prodrugs to toxic species (E. coli cytosine deaminase which converts 5FC to 5FU); and 4) stimulate immune responses (costimulatory molecules B7.1 and B7.2). The effects of these vectors are being investigated in several tumor models. We have examined tumor suppressor gene expression and function in normal and transformed mammary epithelial cells. We found that some drug resistant MCF-7 sublines retained wild type p53 expression and function while others developed an intermediate G1 arrest (despite having wild type p53) and some developed mutations and had no G1 arrest following radiation. These studies suggest that drug resistant cells may develop downstream defects that overcome the inhibitory effects of p53 following DNA damage. We found that BRCA1 RNA levels increased during G1/S and were induced in MCF-7 breast cancer cells following treatment with estradiol. We isolated a VP-16 resistant cell line which overexpresses the multidrug resistance associated protein (MRP) and found that a mitoxantrone resistant cell line was cross-resistant to topoisomerase I inhibitors. Resistance in this cell line is associated with decreased drug accumulation in the absence of MDR1 or MRP expression. Breast cancer clinical trials investigated the role of cytokines in ameliorating hematopoietic toxicities of dose intensive chemotherapy. We found that sequential administration of IL-3 and GM-CSF with FLAC chemotherapy was associated with less platelet toxicity than GM-CSF alone. Studies by Drs. Schwartz and Gress suggested that the toxic effects of chemotherapy and cytokines on progenitor cells may be mediated through negative regulators of hematopoiesis. We initiated trials of a bispecific antibody which binds both erbB2 receptors on tumor cells and FcgRI receptors on monocytes and granulocytes. In collaboration with NHLBI, we have three clinical trials exploring gene transfer into hematopoietic progenitor cells (HPCs) in patients with metastatic breast cancer.
